New insights are provided into the mechanism of interaction between Trichoderma and …

• Abstract
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• Results
• Discussion and Conclusion
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Biology Articles » Mycology » Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride » Results

Results

- Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride

Trichoderma metabolite mixtures activate a Ca²⁺-mediated signalling in soybean cells

Fungal culture filtrates obtained from T. atroviride strain P1 wild type were tested on soybean cells stably expressing in the cytosol the bioluminescent Ca²⁺ indicator aequorin. In the Ca²⁺ measurement experiments fungal metabolite mixtures were applied to cells at a dose (4-fold concentrated culture medium) corresponding to that commonly used for in vivo bioassays of physiological effects (i.e. ISR and elicitor activity) on plants [10]. In preliminary dose-response experiments, the above concentration was found to induce about half of the maximum effect on [Ca²⁺]_cyt increase (data not shown). The whole culture filtrate of Trichoderma elicited a strong Ca²⁺ elevation that was generated without an evident lag phase after the metabolite mixture application. A Trichoderma "Ca²⁺ signature" could be identified, which was characterized by a maximum of [Ca²⁺]_cyt (6.09 ± 0.11 μM), reached after about 1 min, followed by a decrease within 20 min to 0.75 ± 0.06 μM, without returning to resting values (~100 nM) (Fig. 1a). No [Ca²⁺]_cyt change was observed in control cells treated with the non-inoculated fungal culture medium (Fig. 1a). The Trichoderma metabolites were fractionated by using a 3 kDa cut-off and the two separated fractions were applied to soybean cells. The resulting Ca²⁺ transients showed, after a first Ca²⁺ peak nearly superimposable in time, very different kinetic trends characterized by a slow and modulated pattern of signal dissipation with the >3 kDa fraction, and a rapid decline of the Ca²⁺ concentration to the basal level with the 1b). The combination of these two Ca²⁺ traces plus a plausible synergistic effect of the molecular components of the two fractions may account for the kinetics of the Ca²⁺ change observed with the unfractionated metabolite mixture (Fig. 1a).

In order to determine whether the Trichoderma Ca²⁺ signature is modified when the fungus is cultured with the pathogen B. cinerea, we tested metabolite mixtures produced by B. cinerea grown alone and during the coculture of these two fungi. Size-fractionated culture filtrates from the pathogenic fungus triggered in soybean cells Ca²⁺ changes characterized by special features, such as an exceptionally high Ca²⁺ elevation (7.53 ± 0.15 μM) caused by the 2+ level recorded with both μ

M) and >3 kDa (0.47 ± 0.03 μM) fractions (Fig. 1c). The Ca²⁺ transients observed upon cell treatment with both the fractions derived from Trichoderma cultured in the presence of Botrytis showed a single main Ca²⁺ peak occurring at different time values and, with the 1d). It is noteworthy that different kinetics of the Ca²⁺ signals were generated in soybean cells by the co-application of the filtrates (both >3 and 1d, inset) in comparison to the traces induced by those of the cocultured fungi (Fig. 1d). These results suggest that the antagonism condition modifies the quality/quantity of the molecules accumulated in the culture media and indicate that the presence of the phytopathogenic host may significantly affect the Ca²⁺ response of plant cells to Trichoderma. The lack of a Trichoderma specific endochitinase modifies the kinetics of the Ca2+ changes

The >3 kDa metabolite mixture from an endochitinase gene knock-out Trichoderma mutant, unable to produce the 42 kDa endochitinase (CHIT42) [10], induced a Ca²⁺ transient clearly different from that of the wild type, both in the occurrence and level of the peaks. In addition, the Ca²⁺ signal dissipated almost completely within 10 min (Fig. 2a, compare with Fig. 1b). These findings suggest that CHIT42 is among the Trichoderma metabolites that may be perceived as elicitor by plant cells, and is likely to account for the sustained [Ca²⁺]_cyt level over the time. The Trichoderma Δech42 mutant grown alone induced a Ca²⁺ trace that did not significantly differ from the wild type (Fig. 2a, compared with Fig. 1b). On the other hand, when the Δech42 mutant was cocultivated with B. cinerea, also the 2+ profile quite different from the corresponding wild type fraction and more closely resembling the Botrytis-induced Ca²⁺ change (Fig. 2b, compared with Fig. 1c, d).

Trichoderma metabolite mixtures elicit defense reactions in plant cells

Intracellular ROS accumulation

One of the earliest plant responses at the cellular level to fungal pathogen infection is an increased production of intracellular ROS [11]. Preliminary tests indicated that a time interval between 5 and 10 min after the treatment was optimal to measure intracellular ROS accumulation by using dichlorofluorescein diacetate (DCF) [12] (data not shown). Compared to control cells, that showed no fluorescence at all (Fig 3a'), both >3 and Trichoderma metabolite fractions induced a faint detectable signal (Fig. 3b' and 3f'). As expected in the case of a necrotrophic pathogen, Botrytis filtrates, mainly 3c' and 3g') than that of the biocontrol agent. ROS accumulation was very low when metabolites from Trichoderma cocultured with Botrytis were applied (Fig. 3d' and 3h'). In particular, in the case of the Trichoderma+Botrytis fraction (Fig. 3h') the significant reduction in DCF fluorescence may be attributed to the high percentage of dead cells (60.4 ± 1.8 % after 10 min) (see also below). No evident differences were found when cells were treated with filtrates of the Δech42 mutant compared to the wild type (see for example Fig. 3e'), unless the Δech42 + Botrytis was applied (Fig. 3i', compared with 3h'). These findings indicate that, besides the generation of specific Ca²⁺ signatures, other processes are differentially affected by metabolites secreted by the phytopathogen and the biocontrol agent. It can be speculated that the induction of ROS does not play a major role in the plant cell response to Trichoderma metabolite mixtures.

Reduction in cell viability

Intracellular Ca²⁺ overload may determine cytotoxicity and cause either apoptotic or necrotic cell death [13]. In view of the high levels of [Ca²⁺]_cyt induced by some of the fungal culture filtrates, their effect on cell viability was determined. Based on Evans Blue staining, all metabolite mixtures significantly increased after 30 min the percentage of dead cells in comparison with untreated controls, except the >3 kDa culture filtrate from the Δech42 mutant (Fig. 4). The reduction in cell viability was more remarkable with 4b) than >3 kDa (Fig. 4a), suggesting a major toxic effect played by low MW metabolites.

Induction of programmed cell death

Detection of caspase activation, a strictly PCD-related event ([14] and references herein), was used to determine whether cell death induced by the fungal metabolite mixtures occurred via PCD rather than a necrotic event. In soybean control cells a low level of caspase 3-like activity, measured by quantification of free p-nitroaniline (0.018 ± 0.002 mM pNA), and probably due to normal cell turnover, was detected (Fig. 5a). In agreement with the results of the cell viability test, a significant increase of caspase 3-like protease activity was caused by 30 min application of both >3 and Trichoderma wild type grown alone (Fig. 5a and 5b). This indicates that PCD is part of the plant cell response to Trichoderma metabolites. Interestingly, the Trichoderma-Botrytis coculture, although generating the maximal cell death percentage (Fig. 4b), was not found to trigger a significant caspase 3-like activation (Fig. 5b), suggesting the induction of a different mode of cell death.

When cells were treated with the corresponding fraction of the Δech42 Trichoderma mutant cocultured with Botrytis, a statistically significant activity of caspase 3-like protease was recorded, and this value (0.038 ± 0.004 mM pNA) approached that obtained with the Botrytis filtrate (0.034 ± 0.003 mM pNA). In all experiments, the addition of a caspase 3 specific inhibitor (Ac-DEVD-CHO) lowered the amount of free pNA released from the substrate to the level of the control (data not shown), thus confirming the validity of the test for caspase 3-like activity.

Chromatin condensation and morphological cell alterations

Hoechst 33342 (HO)/Propidium Iodide (PI) staining followed by morphological analysis provided additional information on the type of cell death caused by the fungal metabolite mixtures produced in all the considered experimental conditions. Fig. 5c shows the staining pattern of soybean cells incubated with the plasma membrane-permeable DNA-binding agent HO and with the impermeant dye PI in the presence or absence of the 5d). Cells treated with filtrates from Trichoderma wild type grown alone showed the prevalence of HO positive/PI negative nuclei, indicative of early PCD-like stages, characterized by chromatin condensation and an intact plasma membrane (Fig. 5c). Ultrastructural analyses confirmed these findings, showing small lumps of condensed chromatin close to an intact nuclear envelope and plasma membrane stuck to the cell wall in about 70% of the cells (Fig. 5d).

Most cells incubated with Botrytis metabolite mixture were HO positive/PI positive (late PCD stages), with both condensed chromatin and a functionally altered plasma membrane (Fig. 5c). Electron microscope observations indicated an evident chromatin condensation just beneath the nuclear envelope, chloroplasts and mitochondria altered in their organization, and plasma membrane detached from the cell wall (Fig. 5d).

The HO negative/PI positive staining pattern obtained with the 5c) revealed the absence of chromatin condensation and the breakdown of the plasma membrane, both characteristics of a necrotic status of the cells. The induction of a necrotic pathway was also supported by the lack of caspase 3-like activation (Fig. 5b). In the majority of the cells, the ultrastructure appeared deeply affected, with the plasma membrane completely detached and nuclei with a disorganized or absent nucleoplasm and heterogeneous residual chromatin clumps. Chloroplasts and mitochondria looked remarkably altered in their structure (Fig. 5d). Cell necrosis was somehow expected since during antagonism/mycoparasitism T. atroviride strain P1 typically releases trichorzianines, [15]. However, both the induction and effectiveness of these antibiotics require the action of endochitinases and other cell wall degrading enzymes on the host tissues, which explains the results obtained when the ech42 deletion mutant was used in the coculture instead of the wild type. In this case, the lack of the major chitinase activity may have reduced the induction and accumulation of the necrogenic metabolites, which resulted in a typical late PCD-like staining (HO positive/PI positive) (Fig. 5c). This is confirmed by electron microscope observations, which likewise suggest a change in the induced cell death pathway (necrosis versus PCD) when wild type Trichoderma is replaced by the mutant strain in the dual coculture (Fig. 5d). No ultrastructural differences were found between treatments with Trichoderma wild type or Δech42 mutant grown alone (Fig. 5d).